From 99eafb7530451efd234dddb059fda06aaf8bf3d2 Mon Sep 17 00:00:00 2001 From: Lorenzo Zolfanelli Date: Sun, 11 Oct 2020 16:27:28 +0200 Subject: [PATCH] incluse correzioni Capitans CAP 2 --- chapters/2-setup.tex | 435 +- images/microscope.pdf | Bin 40690 -> 66165 bytes images/setup.pdf | Bin 0 -> 159052 bytes images_src/microscope.svg | 5052 ++++++++++---- images_src/setup.svg | 13126 ++++++++++++++++++++++++++++++++++++ 5 files changed, 17009 insertions(+), 1604 deletions(-) create mode 100644 images/setup.pdf create mode 100644 images_src/setup.svg diff --git a/chapters/2-setup.tex b/chapters/2-setup.tex index 8fa27f3..3d41005 100644 --- a/chapters/2-setup.tex +++ b/chapters/2-setup.tex @@ -5,7 +5,8 @@ L'apparato sperimentale realizzato consiste in un microscopio invertito, progettato con la collaborazione dell'Officina Meccanica del LENS, che combina l'illuminazione a luce trasmessa con un sistema -di due pinzette ottiche posizionabili elettronicamente e con uno +di due pinzette ottiche posizionabili attraverso deflettori acusto-ottici +e con uno schema di microscopia di epifluorescenza, in grado di raggiungere la sensibilità di singola molecola e di alternare diversi schemi di illuminazione (campo largo, TIRF, HILO). @@ -16,22 +17,22 @@ Una rappresentazione schematica del microscopio è mostrata in figura %%%%%%%%% %%%%%%%%% %%%%%%%%% %%%%%%%%% %%%%%%%%% %%%%%%%%% %%%%%%%%% Il sistema è ottimizzato per consentire la coesistenza e il -funzionamento simultaneo - senza interferenze - dei tre principali +funzionamento simultaneo - minimizzando le sovrapposizioni +spettrali - dei tre principali schemi di microscopia e manipolazione: \begin{description} \item[Illuminazione a luce trasmessa:] la luce emessa da una - sorgente LED a spettro largo viene collimata da un + sorgente LED viene collimata da un condensatore e trasmessa dall'alto verso il basso attraverso il campione. L'obiettivo posto sotto il campione raccoglie la luce - trasmessa e ricostruisce un'immagine ingrandita del - contrasto di densità dello stesso. + trasmessa e ricostruisce un'immagine ingrandita dello stesso. \item[Pinzette ottiche:] due intensi fasci laser vengono collimati sul piano focale posteriore dell'obiettivo, quindi focalizzati sul campione. In questo modo possono essere usati per intrappolare e manipolare microsfere - in soluzione. La radiazione laser diffusa che attraversa + in soluzione. La radiazione laser che attraversa il campione e viene raccolta dal condensatore può essere analizzata per monitorare lo stato delle trappole e lo spostamento dal centro delle sfere intrappolate. @@ -52,9 +53,9 @@ schemi di microscopia e manipolazione: \label{fig:microscope} \end{figure} -Per evitare interferenze tra i tre schemi è stato necessario -scegliere opportune lunghezze d'onda per la radiazione di -illuminazione e, di conseguenza, appropriati specchi dicroici +Per evitare interferenze è stato necessario +scegliere opportune lunghezze d'onda per i tre schemi ottici +e, di conseguenza, appropriati specchi dicroici e filtri. L'illuminazione a luce trasmessa è realizzata sfruttando una @@ -66,6 +67,37 @@ laser a diodo alle lunghezze d'onda di \SIlist{488;532;635}{\nm} e l'emissione dei fluorofori fino a \SI{700}{\nm} può essere raccolta e disaccopiata dall'illuminazione a luce trasmessa. +Tuttavia, la necessità di ottenere una sensibilità di singola molecola +rende particolarmente critica la scelta delle caratteristiche +spettrali e di qualità delle sorgenti e delle componenti ottiche +utilizzate. In particolare, come vedremo nel dettaglio in sezione +\ref{sec:stabilization}, la stabilizzazione meccanica del sistema +richiede un’illuminazione a luce trasmessa del campione di intensità +sufficiente a produrre una chiara immagine delle microsfere +immobilizzate sui sensori CMOS. Allo stesso tempo, la microscopia di +fluorescenza richiede la rilevazione di segnali estremamente deboli, +attraverso un sensore di tipo EmCCD raffreddato estremamente +sensibile. In questo contesto, anche il passaggio di pochi fotoni al +secondo tra i due cammini ottici comprometterebbe la sensibilità del +sistema. Per risolvere questo problema è state scelta una sorgente LED +con un profilo di emissione sufficientemente lontano dalle lunghezze +d’onda usate per la fluorescenza (400-700 nm) e il posizionamento di +un filtro ad alta efficienza per per eliminare l’emissione residua del +LED nella regione di sovrapposizione. Un altra serie di filtri ad +altra efficienza è stata posta in prossimità del sensore EMCCD, +procendendo anche alla completa separazione spaziale (attraverso tubi, +diaframmi e separatori) del cammino ottico, in modo eliminare quasi +completamente la componente di radiazione LED che raggiunge la +camera. L’utilizzo di un LED con emissione centrata nel vicino +infrarosso, con lunghezza d’onda sensibilmente più vicina al diametro +delle microsfere utilizzate, ha inoltre effetti significativi sul +profilo radiale di intensità delle immagini prodotte delle +microsfere. Questa differenza, rispetto ai profili osservati con un +illuminazione alle lunghezze d’onda del visibile, ha reso necessario +lo sviluppo ex-novo di un algoritmo per la determinazione della +posizione del piano focale rispetto al centro della sfera, descritto +sempre in sezione \ref{sec:stabilization}. + \section{Microscopio e traslatori} @@ -90,10 +122,12 @@ Per gli scopi di questa tesi sono stati usati due obiettivi differenti: \begin{itemize} - \item Un obiettivo planapocromatico 60x a immersione ad acqua, - con alta apertura numerica ($NA = 1.22$) e distanza di lavoro - di \SI{0.2}{\mm} (Nikon) - \item Un obiettivo TIRF (??) a immersione a olio (Nikon) + \item Un obiettivo planapocromatico 60x a immersione ad acqua, con + alta apertura numerica ($NA = 1.22$) e distanza di lavoro di + \SI{0.2}{\mm} (Nikon) + \item Un obiettivo TIRF 60x a immersione a olio, con alta apertura + numerica ($NA = 1.49$) e distanza di lavoro di \SI{0.13}{\mm} + (Nikon). \end{itemize} Il primo obiettivo presenta le caratteristiche migliori per quanto @@ -104,18 +138,19 @@ illuminazione TIRF, al prezzo di un'efficienza delle pinzette ottiche che decresce rapidamente all'aumentare del volume di campione attraversato. -La posizione del campione rispetto al centro del percorso ottico -può essere modificata attraverso due traslatori controllati -elettronicamente, uno di tipo piezoelettrico (Physik Instrumente, -P-527) -per spostamenti veloci e con risoluzione nanometrica, con una corsa di -\SI{100}{\um} per asse, e uno di tipo motore passo-passo -(Steinmeyer, KDT105-PM) per spostamenti macroscopici con una corsa di \SI{50}{\mm} per asse. +La posizione del campione rispetto al centro del percorso ottico può +essere modificata attraverso due traslatori controllati +elettronicamente, uno, il traslatore a corto raggio, di tipo +piezoelettrico (Physik Instrumente, P-527) per spostamenti veloci e +con risoluzione di \SI{1}{\nm}, con una corsa di \SI{100}{\um} per +asse, e l'altro, il translatore a lungo raggio, dotato di un motore +piezoelettrico (Steinmeyer, KDT105-PM) per spostamenti macroscopici con +una corsa di \SI{50}{\mm} per asse e risoluzioni di \SI{100}{\nm}. Il piano focale può essere modificato variando la posizione dell'obiettivo, grazie a un posizionatore verticale piezoelettrico (Physik Instrumente, PIFOC™ P-725) con con una corsa di \SI{400}{\um} -e risoluzione nanometrica. Il microscopio è stato progettato in modo +e risoluzione di \SI{2}{\nm}. Il microscopio è stato progettato in modo che al centro della corsa il piano focale si trovi in corrispondenza della superficie interna del vetrino portaoggetti (per un campione preparato nel modo descritto nei paragrafi precedenti). @@ -144,50 +179,53 @@ manipolazione del campione particolarmente semplice e intuitiva. \section{Illuminazione a luce trasmessa} La radiazione emessa da un LED ad alta intensità (Thorlabs, M780L3) -con picco di emissione centrato nell'infrarosso, a \SI{780}{nm}, -viene filtrata con un filtro passa-basso (Thorlabs, FEHL0750) in modo -da sopprimere l'emissione a lunghezze d'onda inferiori a \SI{750}{nm} -che andrebbe a interferire con il rilevamento della fluorescenza. +con picco di emissione centrato nell'infrarosso, a \SI{780}{nm}, viene +filtrata con un filtro passa-basso (Thorlabs, FEHL0750) in modo da +sopprimere l'emissione a lunghezze d'onda inferiori a \SI{750}{nm} che +andrebbe a interferire con il rilevamento della fluorescenza. Successivamente, con l'ausilio di una singola lente e un accoppiatore -in fibra (Thorlabs), la radiazione emessa dal LED e filtrata -viene immessa in una fibra ottica e trasportata in corrispondenza del -microscopio. All'altra estremità della fibra un sistema formato da un -collimatore, un diaframma e una lente aggiusta le dimensioni e la -divergenza del fascio di illuminazione, direzionandolo collimato verso -il piano focale posteriore del condensatore. -Il fascio di illuminazione viene quindi focalizzato sul campione, lo -attraversa e la radiazione trasmessa e diffusa viene raccolta -dall'obiettivo. -Gli obiettivi usati sono corretti all'infinito, questo significa -che i raggi in uscita dall'obiettivo sono collimati. -Inserendo nel percorso ottico una \textit{lente di tubo} l'immagine -viene messa a fuoco in un piano ben preciso. -La distanza focale dell'obiettivo è di \SI{3.3}{\mm}, quella della -lente di tubo di \SI{250}{\mm}. -Otteniamo quindi la formazione di un immagine del piano del campione -selezionato a una distanza di \SI{250}{\mm} dalla lente e con un -fattore di ingrandimento $M = 250/3.3 \approx 75$. +in fibra (Thorlabs), la radiazione emessa dal LED e filtrata viene +immessa in una fibra ottica e trasportata in corrispondenza del +microscopio. All'altra estremità della fibra, in basso a destra nello +schema ottico mostrato in figura \ref{fig:setup}, un sistema formato +da un collimatore, un diaframma e una lente (\texttt{L-BF} nello +schema) aggiusta le dimensioni e la divergenza del fascio di +illuminazione, focalizzandolo sul piano focale posteriore del +condensatore. Il fascio di illuminazione collimato illumina il +campione, lo attraversa e la radiazione trasmessa e diffusa viene +raccolta dall'obiettivo. Gli obiettivi usati sono corretti +all'infinito, questo significa che le aberrazioni sono corrette quando +il campione è posto sul piano focale anteriore dell'obiettivo e i +raggi provenienti da tale piano e in uscita dall'obiettivo sono +collimati. Inserendo nel percorso ottico una \textit{tube lens} +(\texttt{L-Im1}), posta dopo il dicroico di fluorescenza +(\texttt{DIC-Fluor}), l'immagine viene quindi formata sul piano focale +(\texttt{Image plane}) della stessa lente. La distanza focale +dell'obiettivo è di \SI{3.3}{\mm}, quella della \textit{tube lens} di +\SI{250}{\mm}. Otteniamo quindi la formazione di un'immagine del +piano del campione selezionato a una distanza di \SI{250}{\mm} dalla +lente e con un fattore di ingrandimento $M = 250/3.3 \approx 75$. Questa immagine viene ulteriormente ingrandita mediante l'uso di -un'ulteriore lente con distanza focale \SI{75}{\mm} posta a -\SI{350}{mm} dalla lente di tubo. -In questo modo si ottiene, a una distanza di \SI{30}{\cm} dall'ultima -lente, un ulteriore ingrandimento di un fattore -$M' = 300 / (350-250) = 3$. -A questa distanza l'immagine viene -rilevata, dopo aver diviso il cammino ottico in due rami con un -\textit{beam-splitter}, da due sensori CMOS monocromatici -(Thorlabs DCC1545M). -La potenza viene suddivisa tra i due sensori secondo il rapporto -90:10. Il sensore che riceve la frazione maggiore di potenza sarà -utilizzato per il sistema attivo di stabilizzazione meccanica (descritto -in sezione \ref{sec:stabilization}), l'altro sensore verrà utilizzato -per mostrare costantemente su un monitor la situazione del campione. +un'ulteriore lente (\texttt{L-Im2}) con distanza focale \SI{75}{\mm} +posta a \SI{350}{mm} dalla lente di tubo. In questo modo si ottiene, +a una distanza di \SI{30}{\cm} dall'ultima lente, un ulteriore +ingrandimento di un fattore $M' = 300 / (350-250) = 3$. A questa +distanza l'immagine viene rilevata, dopo aver diviso il cammino ottico +in due rami con un \textit{beam-splitter} (\texttt{BS-BF}), da due +sensori CMOS monocromatici (Thorlabs DCC1545M). La potenza viene +suddivisa tra i due sensori secondo il rapporto 90:10. Il sensore che +riceve la frazione maggiore di potenza sarà utilizzato per il sistema +attivo di stabilizzazione meccanica (descritto in sezione +\ref{sec:stabilization}), dopo aver filtrato la radiazione residua a +\SI{1064}{\nm} con un apposito filtro (\texttt{F-BlockTwz}); l'altro +sensore verrà utilizzato per mostrare costantemente su un monitor +l'immagine del campione. I sensori CMOS usati hanno una risoluzione di \SI{1280x1024}{pixel} e un'area attiva di \SI{6.66x5.32}{\mm}, corrispondenti a una dimensione -di \SI{5.2x5.2}{\um} per ogni pixel. La massima risoluzione temporale -per acquisire un'immagine completa è di \SI{30}{fps}, ma è possibile -ottenere risoluzioni più elevate riducendo la dimensione della regione +di \SI{5.2x5.2}{\um} per ogni pixel. La velocità di acquisizione è di +\SI{30}{fps} per l'intera superficie del sesnore, ma è possibile +incrementarla riducendo la dimensione della regione d'interesse. Tenendo conto del fattore complessivo di ingrandimento @@ -198,42 +236,49 @@ I valori corretti possono essere determinati solo sperimentalmente dopo aver fissato l'allineamento del percorso ottico e delle telecamere. -Per ottenere i fattori reali di conversione tra pixel e dimensioni -sul campione è stato sviluppato un programma in ampiente LabVIEW che, -sfruttando una microsfera immobilizzata sul vetrino portaoggetti, -consente di eseguire una calibrazione assistita. +Per ottenere i fattori reali di conversione tra pixel e dimensioni sul +campione ho sviluppato un programma in ampiente LabVIEW che, +sfruttando una microsfera immobilizzata sul vetrino portaoggetti, +consente di eseguire una calibrazione assistita (sezione +\ref{sec:stabilization}). \section{Pinzette ottiche} \label{sec:tweezer} Il fascio \textit{laser} collimato in uscita da una sorgente -\ce{Nd}:\ce{YVO4} attraversa immediatamente una serie di due isolatori +\ce{Nd}:\ce{YVO4}, in alto a destra nello schema di figura +\ref{fig:setup}, dopo essere stato ridotto in diametro +grazie a un telescopio (lenti \texttt{L-TS4a} e \texttt{L-TS4b}), +attraversa immediatamente una serie di due isolatori ottici, necessari per ridurre al minimo le instabilità della sorgente -dovute alla retro-riflessione. +dovute alla retro-riflessione, e viene nuovamente ingrandito con un +secondo telescopio (lenti \texttt{L-TS5a} e \texttt{L-TS5b}). Successivamente, dopo aver attraversato una lamina $\lambda/2$, -viene diviso in due fasci ortogonali da un cubo polarizzatore. +viene diviso da un \textit{beam splitter} polarizzatore (\texttt{BS-Twz1}) +in due fasci ortogonali. I due fasci avranno polarizzazioni parallele o perpendicolari rispetto alla superficie del tavolo ottico, e la lamina $\lambda/2$ consente di modificare la ripartizione di potenza tra i due rami. I due rami vengono ricombinati utilizzando un secondo cubo -polarizzatore, dopo aver attraversato due modulatori acusto-ottici -(\textit{Acousto Optic Modulator, AOM}). -Gli AOM vengono realizzati ponendo un cristallo di diossido +polarizzatore (\texttt{BS-Twz2}), dopo aver attraversato due +deflettori acusto-ottici +(\textit{Acousto Optic Deflector, AOD}). +Gli AOD vengono realizzati ponendo un cristallo di diossido di tellurio (\ce{TeO2}) in contatto con un trasduttore piezoelettrico: la radiofrequenza trasmessa al trasduttore consente l'instaurazione di un onda stazionaria acustica all'interno del cristallo, che si comporta sostanzialmente come un reticolo di diffrazione. Variando l'ampiezza e la frequenza del segnale -elettrico inviato agli AOM è possibile modificare le caratteristiche +elettrico inviato agli AOD è possibile modificare le caratteristiche dell'onda acustica stazionaria nel cristallo, e quindi ottenere una -deflessione e una modulazione del fascio in uscita. +deflessiondel fascio in uscita. -Gli elementi ottici sono allineati in modo che quanto a entrambi -gli AOM viene applicata una radiofrequenza di \SI{75}{MHz} i due rami +Gli elementi ottici sono allineati in modo che quando a entrambi +gli AOD viene applicato un segnale di \SI{75}{MHz} i due rami incidano perpendicolarmente sull'ultimo cubo polarizzatore e proseguano -il loro percorso esattamente sovrapposti (linea continua nella +il loro percorso esattamente sovrapposti (linea continua nella figura \ref{fig:aom}). \begin{figure}[ht] @@ -245,9 +290,9 @@ figura \ref{fig:aom}). \end{figure} Una coppia di lenti piano-convesse incrementa la dimensione del fascio -e rende il +e rende il piano focale posteriore dell'obiettivo coniugato con il piano -delle uscite degli AOM. In questo modo, per qualsiasi deflessione +delle uscite degli AOD. In questo modo, per qualsiasi deflessione angolare introdotta sui due rami i fasci delle due trappole passeranno sempre collimati per il centro del piano focale posteriore dell'obiettivo, con angolo d'incidenza dipendente dalla radiofrequenza @@ -256,43 +301,51 @@ focalizzati dentro il campione propagandosi parallelamente all'asse ottico principale. La variazione di angolo di incidenza nel piano focale posteriore dell'obiettivo si tradue in uno spostamento orizzontale dei fasci nel piano del campione: in questo modo, -regolando la radiofrequenza degli AOM è possibile spostare la posizione +regolando la radiofrequenza degli AOD è possibile spostare la posizione delle trappole lungo una linea orizzontale all'interno del campione. I fattori di conversione tra posizione della trappola nel campione e -frequenza inviata all'AOM, determinati dalle caratteristiche degli AOM +frequenza inviata all'AOD, determinati dalle caratteristiche degli AOD e dalla geometria del sistema ottico, vengono determinati a posteriori -visualizzando microsfere intrappolate e creando rette di calibrazione. - -I modulatori acusto-ottici utilizzati (AA Opto-Electronic, DTSXY-250) -sfruttano onde acustiche di taglio e richiedono una polarizzazione -in ingresso lineare e perpendicolare all'onda acustica. -Per questo nel percorso ottico vengono aggiunte due ulteriori -lamine $\lambda / 2$: una permette di aver per entrambi gli AOM la -stessa polarizzazione in ingresso, la seconda ruota nuovamente di -\SI{90}{\degree} la polarizzazione di uno dei due fasci in modo che -abbiano polarizzazione ortogonali prima di ricombinarsi. -L'intervallo di scansione angolare consentito da questi AOM è di circa -\SI{2.8}{\degree}, corrispondenti a variazioni di \SI{50}{\MHz} nella -radiofrequenza fornita. Lungo questo intervallo l'efficienza dell'AOM, -ovvero la frazione di fascio deflessa nel primo ordine di diffrazione, -può variare significativamente. Questo dipendenza può essere nociva -per gli esperimenti con le pinzette ottiche: se l'intensità -dei fasci non è uniforme per tutte le posizioni della trappola, -la rigidità di essa varierà durante gli spostamenti, rendendo -difficile la manipolazione del campione attraverso lunghe distanze e -potenzialmente falsando i risultati degli esperimenti. -Variando l'angolo di incidenza del fascio rispetto all'AOM è possibile -ottimizzare questa dipendenza intorno a un intervallo di deflessioni -angolari di nostro interesse. Resta comunque una variazione non -trascurabile della rigidità delle trappole in funzione della posizione -e per tenerne conto si utilizza un'apposita procedura di calibrazione -in cui la rigidità viene misurata osservando la diffusione di una -microsfera nella trappola, per una griglia equispaziata di punti. +visualizzando microsfere intrappolate e creando rette di calibrazione +(vedi sezione \ref{sec:calibration}). + +I deflettori acusto-ottici utilizzati (AA Opto-Electronic, DTSXY-250) +sfruttano onde acustiche di taglio, in cui la velocità di propagazione +dell'onda è perpendicolare al fascio in ingresso; in questo modo i +nodi dell'onda stazionaria si dispongono lungo un asse parallela al +banco ottico. +Questo tipo di deflettori richiedono una polarizzazione in +ingresso lineare e perpendicolare all'onda acustica e producono in uscita un fascio con polarizzazione ortogonale rispetto a quella in ingresso. +Per questo nel +percorso ottico vengono aggiunte due ulteriori lamine $\lambda / 2$: +una permette di aver per entrambi gli AOD la stessa polarizzazione in +ingresso, la seconda ruota nuovamente di \SI{90}{\degree} la +polarizzazione di uno dei due fasci in modo che abbiano polarizzazione +ortogonali prima di ricombinarsi. L'intervallo di scansione angolare +consentito da questi AOD è di circa \SI{2.8}{\degree}, corrispondenti +a variazioni di \SI{50}{\MHz} nella radiofrequenza fornita. Lungo +questo intervallo l'efficienza dell'AOD, ovvero la frazione di fascio +deflessa nel primo ordine di diffrazione, può variare +significativamente. Questa dipendenza può essere nociva per gli +esperimenti con le pinzette ottiche: se l'intensità dei fasci non è +uniforme per tutte le posizioni della trappola, la costante elastica +della pinzetta ottica varierà durante gli spostamenti, rendendo difficile la manipolazione +del campione attraverso lunghe distanze e potenzialmente falsando i +risultati degli esperimenti. Variando l'angolo di incidenza del +fascio rispetto all'AOD è possibile ottimizzare questa dipendenza +intorno a un intervallo di deflessioni angolari di nostro +interesse. Resta comunque una variazione non trascurabile della +rigidità delle trappole in funzione della posizione e per tenerne +conto si utilizza un'apposita procedura di calibrazione in cui la +rigidità viene misurata osservando la diffusione di una microsfera +nella trappola, per una griglia equispaziata di punti +(procedura descritta in sezione \ref{sec:calibration}). Per poter osservare e registrare gli spostamenti delle microsfere nella trappole così realizzate è stato implementato un sistema -di rilevamento della radiazione diffusa nel piano focale posteriore +di rilevamento della radiazione nel piano focale posteriore del +condensatore (\textit{back-focal plane detection}, BFPD). \begin{figure}[ht] @@ -318,7 +371,7 @@ polarizzatore, e inviate rispettivamente a due fotodiodi a quadranti coniugato con il piano focale posteriore del condensatore. In questo piano la distribuzione spaziale del campo elettrico è data dalla trasformata di Fourier del campo in corrispondenza del piano -del campione messo a fuoco. È possibile dimostrare che nel caso di +del campione messo a fuoco. È possibile dimostrare che nel caso di una sfera spostata rispetto al centro della trappola l'asimmetria del profilo d'intensità nel piano focale posteriore sarà direttamente proporzionale allo spostamento della sfera rispetto al centro @@ -340,7 +393,7 @@ spostamento della microsfera. \end{figure} Maggiori dettagli sull'elettronica che consente il controllo -della deflessione attraverso gli AOM e la lettura del segnale +della deflessione attraverso gli AOD e la lettura del segnale dei QPD sono forniti nell'appendice \ref{app:electronics}, mentre le procedure di calibrazione sono trattate nella sezione \ref{sec:calibration}. @@ -383,30 +436,46 @@ tramite tre collimatori con specifiche diverse. \caption{Collimatori usati per estrarre i fasci alle varie lunghezzed d'onda, con distanza focale indicata dal costruttore (f) e diametro del fascio (BD) misurato mediante \textit{fit} - gaussiano del suo profilo d'intensità.} + gaussiano del suo profilo d'intensità. Nella colonna + M è riportato il fattore d'ingrandimento che abbiamo selezionato + per ottenere fasci con dimensioni consistenti, attraverso l'utilizzo + di telescopi.} \label{tab:my_label} \end{table} -I fasci a \SIlist{488;635}{\nm} vengono ingranditi mediante -due telescopi realizzati con lenti piano-convesse. -L'emissione dei laser a diodo deve essere filtrata attraverso -filtri passa-banda stretti per evitare che radiazione a lunghezze -d'onda spurie diffonda nel campione e contamini l'emissione di -fluorescenza. - -I tre fasci laser vengono combinati in un unico percorso ottico e -ingranditi di un fattore 10x per mezzo di un ulteriore telescopio -sul percorso comune, raggiungendo un diametro $\approx \SI{2}{\cm}$. +I fasci a \SIlist{488;635}{\nm} vengono ingranditi mediante due +telescopi realizzati con lenti piano-convesse, in questo modo si +ottengono dimensioni del fascio, e quindi caratteristiche di +uniformità nell'illuminazione del campione, confrontabili e simili per +tutte le lunghezze d'onda usate. Per il fascio a 635 nm si ottiene un +fattore d’ingrandimento di 1.66x utilizzando le lenti \texttt{L-TS1a} +e \texttt{L-TS1b}, per quello a \SI{488}{\nm} un fattore +d’ingrandimento 3.33x utilizzando le lenti \texttt{L-TS2a} e +\texttt{L-TS2b}. L'emissione dei laser a diodo deve essere filtrata +attraverso filtri passa-banda stretti per evitare che radiazione a +lunghezze d'onda spurie diffonda nel campione e contamini l'emissione +di fluorescenza. + +I tre fasci laser vengono combinati in un unico percorso ottico +attraverso due specchi dicroici (uno ottimizzato per riflettere il +90\% della radiazione a lunghezze d'onda inferiori ai 532 nm, +\texttt{DIC-532}, e l'altro per riflettere il 90\% di quelle inferiori +a 488 nm, \texttt{DIC-488}). Successivamente il fascio con le tre +lunghezze d'onda combinate viene ingrandito di un fattore 10x per +mezzo di un ulteriore telescopio sul percorso comune, formato dalle +lenti \texttt{L-TS3a} e \texttt{L-TS3b}, raggiungendo un diametro +$\approx \SI{2}{\cm}$. I fasci combinati sono focalizzati sul piano focale posteriore -dell'obiettivo da una lente con $f = \SI{500}{\mm}$, ottenendo +dell'obiettivo da una lente con focale $f = \SI{500}{\mm}$, ottenendo, +in combinazione con uno dei due obiettivi di focale \SI{3.3}{\mm}, un fattore di scala di circa $1/150$ e quindi un diametro $(\textrm{BD})_{1/e^2}$ sul campione di circa \SI{130}{\um}. Per immettere i fasci di eccitazione nel cammino ottico che porta all'obiettivo si usa un dicroico di fluorescenza a tre bande, in grado di riflettere le tre lunghezze d'onda di eccitazione e trasmettere tre finestre di emissione nel quale ricadono i fluorofori -più comuni. +più comunemenete eccitati a tali lunghezze d'onda. La fluorescenza riemessa all'indietro dal campione, e quindi trasmessa dal dicroico di fluorescenza, è combinata con l'illuminazione a luce trasmessa, da cui sarà disaccoppiata per mezzo di un successivo @@ -417,20 +486,30 @@ condivide la stesse ottiche di ingrandimento con il percorso di raccolta della luce trasmessa e quindi lo stesso fattore di ingrandimento $\approx 225x$. -Uno specchio dicroico con frequenza di taglio di \SI{705}{\nm} +Uno specchio dicroico passa-alto con frequenza di taglio di \SI{705}{\nm} viene utilizzato per estrarre l'emissione di fluorescenza dal cammino di raccolta e indirizzarla verso il sensore di una fotocamera di tipo \textit{Electron-Multiplying Charged Coupled Device (EmCCD)}. La regione attiva del sensore è di \SI{8.19x8.19}{\mm}, -corrispondente ad un quadrato di lato \SI{36}{\um}. Il diametro -del fascio di eccitazione è significativamente maggiore di questo +corrispondente ad un quadrato di lato \SI{36}{\um} sul piano del campione. +Il diametro +del fascio di eccitazione, \SI{130}{\um}, +è significativamente maggiore di questo valore per massimizzare l'uniformità della potenza con la quale la regione di interesse viene illuminata. - -La camera usata per la rilevazione della fluorescenza è una -ImagEM X2 C9100-23B (Hamamatsu) con sensibilità del singolo -fotoelettrone. +Dai calcoli eseguiti in seizone \ref{sec:single_molecule_fluorescence}, +per questi parametri risulta una variazione massima di intensità del +17\% all'interno della regione. + +La camera usata per la rilevazione della fluorescenza è una ImagEM X2 +C9100-23B (Hamamatsu). +Questo tipo di telecamera combina, al funzionamento tipico dei +Charged-Coupled-Device (CCD), uno stadio di moltiplicazione degli +elettroni, che consente di raggiungere, in presenza di raffreddamento +a temperature inferiori ai -60°C, la sensibilità del singolo +fotone. Maggiori dettagli sulla messa in opera del sistema di imaging +sono forniti in sezione \ref{sec:single_molecule_fluorescence}. Per poter realizzare gli schemi di illuminazione HILO e TIRF è stato introdotto, nella parte terminale del percorso di eccitazione, prima @@ -449,7 +528,7 @@ eccitazione in uscita dall'obiettivo. \begin{sidewaysfigure} - \includegraphics[width=1.0\linewidth]{images/Setup.pdf} + \includegraphics[width=1.0\linewidth]{images/setup.pdf} \caption{Vista d'insieme dell'apparato sperimentale} \label{fig:setup} \end{sidewaysfigure} @@ -460,81 +539,81 @@ eccitazione in uscita dall'obiettivo. \toprule Code & Type & Usage & Parameters & Product ID\\ \midrule - \multicolumn{5}{l}{\it Fluorescence Excitation Path}\\ + \multicolumn{5}{l}{\it Percorso eccitazione fluorescenza}\\ F-Clean635 - & Bandpass Filter - & \SI{635}{\nm} laser clean-up + & Filtro passa-banda + & Pulizia spettrale laser \SI{635}{\nm} & $\lambda_{centr}: \SI{640}{\nm}, bw: \SI{14}{\nm}$ & FF01-640/14-25\\ L-TS1a - & Plano-Convex Spherical Lens - & Telescope for \SI{635}{\nm} laser - & f: \SI{0}{\mm} + & Lente sferica piano-convessa + & Telescopio per laser a \SI{635}{\nm} + & f: \SI{30}{\mm} & \\ L-TS1b - & & - & f: \SI{0}{\mm} + & & + & f: \SI{50}{\mm} & \\ L-TS2a - & - & Telescope for \SI{488}{\nm} laser - & f: \SI{0}{\mm} + & + & Telescopio per laser a \SI{488}{\nm} + & f: \SI{30}{\mm} & \\ L-TS2b - & & - & f: \SI{0}{\mm} + & & + & f: \SI{100}{\mm} & \\ DIC-532 - & Single-edge Dichroic Filter - & Excitation beam multiplexing + & Filtro dicroico single-edge + & Inserimento fascio a 532nm & $\lambda_{edge}: \SI{532}{\nm}$ & Di02-R532-25-D\\ DIC-488 - & & + & & Inserimento fascio a 488nm & $\lambda_{edge}: \SI{488}{\nm}$ & Di02-R488-25-D\\ L-TS3a - & Achromatic Doublet - & Telescope for comb. ex. lasers - & f: \SI{0}{\mm} + & Doppietto acromatico + & Ingrandimento fasci fluorescenza + & f: \SI{19}{\mm} & \\ L-TS3b & & - & f: \SI{0}{\mm} + & f: \SI{200}{\mm} & \\ L-Exc & - & Excitation beam focusing - & f: \SI{0}{\mm} + & Focalizzazione fascio fluorescenza + & f: \SI{500}{\mm} & \\ DIC-Fluor - & Quad-edge Dichroic Filter - & Excitation/Emission separation + & Filtro dicroico quad-edge + & Separazione eccitazione/emissione & $\lambda_{edge}: \SIlist{405;488;532;635}{\nm}$ & Di03-R405/488/532/635-t1 \\ - \multicolumn{5}{l}{\it Tweezers Path}\\ + \multicolumn{5}{l}{\it Percorso trappole}\\ L-TS4a - & Plano-Convex Spherical Lens - & Telescope matching isolator size - & f: \SI{0}{\mm} + & Lente sferica piano-convessa + & Inserimento laser 1064nm in isolatore. + & %f: \SI{0}{\mm} & \\ L-TS4b & & - & f: \SI{0}{\mm} + & %f: \SI{0}{\mm} & \\ L-TS5a - & Plano-Convex Spherical Lens - & Telescope after isolator - & f: \SI{0}{\mm} + & Lente sferica piano-convessa + & Ingrandimento fascio 1064nm + & %f: \SI{0}{\mm} & \\ L-TS5b & & - & f: \SI{0}{\mm} + & %f: \SI{0}{\mm} & \\ L-Twz1 - & Plano-Convex Spherical Lens - & Telescope after isolator + & Lente sferica piano-convessa + & Ingrandimento rappole dopo AOD & f: \SI{0}{\mm} & \\ L-Twz2 @@ -542,11 +621,11 @@ eccitazione in uscita dall'obiettivo. & f: \SI{0}{\mm} & \\ DIC-Twz1 - & Single-edge Dichroic Filter - & Tweezer beam insertion + & Lente sferica piano-convessa + & Inserimento fascio trappole & $\lambda_{edge}: \SI{1064}{\nm}$ & \\ - \multicolumn{5}{l}{\it Imaging Path}\\ + \multicolumn{5}{l}{\it Percorso Imaging}\\ L-Im1 & Tube Lens & Tube Lens @@ -558,25 +637,25 @@ eccitazione in uscita dall'obiettivo. & f: \SI{0}{\mm} & \\ DIC-BF - & Single-edge Dichroic Filter - & Fluorescence/Brightfield demux + & Filtro dicroico single-edge + & Seprazione fluorescenza/brightfied & $\lambda_{edge}: \SI{705}{\nm}$ & FF705-Di01-25x36\\ F-BlockBF - & Shortpass Filter - & Residual brightfield suppression + & Filtro passa-basso + & Soppressione brightfield residuo & $\lambda_{cut}: \SI{700}{\nm}$ & FESH0700\\ F-Emiss - & Quad-band bandpass Filter - & Fluorophore emission filtering + & Filtro passa-banda quad-band + & Fluorofori/Soppressione laser eccitazione & $\lambda_{centr}: \SIlist{446;510;581;703}{\nm}$ & FF01-446/510/581/703\\ F-BlockTwz - & Bandstop filter - & Fluorophore emission filtering - & $\lambda_{centr}: \SIlist{446;510;581;703}{\nm}$ - & FF01-446/510/581/703\\ + & Filtro band-stop + & Soppressione laser trappole residuo + & $\lambda_{centr}: 1064nm$ + & \\ \end{tabular} \caption{Lista delle componenti ottiche} \label{tab:optical_components} diff --git a/images/microscope.pdf b/images/microscope.pdf index 5194e5bf259374980abe72d9d43bfa3990cef4e0..d70311115e656ca496a6a217a7087b555f80829d 100644 GIT binary patch delta 51084 zcmV(~K+nJOy#n=w1dvRB?7dB|Ey->rHqT#i#s=Uz0a%{KR^EQUypi~&sy()kE@Q)YkaiNlFLWV zpQGiEfBA=xpT{itGxPt}AAJ47>m`-XcIA)js-NSoAHP24hdyfoC4FAy`uO@N*Zo<} z_A&Bjz7h%Pv-JKk`sdl&B>eWKoS#R*->rX?^4atFxcT2c8P#Gd`Z~ z&$C`Z`T9oThwh1g+bp~Yp6U3wdi!j>eLP62AJ-V4_v}f3KV7r-rF_GW53hw$4r}p7 znlI(gHnuYJXYJ$ZoB6ZMU%Xa&|5&wtY3T!9_O12mGW7A@BlR=Qe%pKGnNrgEIr_bN z1k>ey?vZm=f3LqvSj~IJ=o8iBsZX9rY!W6}6gqKlY5(khq_mz^r?7y%Q=S{EPI>Ks z*(tMg)0{IYwa+u(oTKUUL|yV+5p%xJ+MS7=knYPU`7WR5Tq+5(biVa 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